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Condensation of DNA by monohydric alcohols
Colloids and Surfaces B: Biointerfaces 13 (1999) 157 – 163
Condensation of DNA by monohydric alcohols M. Matzeu a, G. Onori b,*, A. Santucci b b
a Istituto Superiore di Sanita`, Laboratorio di Fisica, V.le Regina Elena, I-00161 Roma, Italy Dipartimento di Fisica, Istituto per la Fisica della Materia, Unita` di Perugia, Uni6ersita` di Perugia, Via A. Pascoli, I-06100 Perugia, Italy
Accepted 3 February 1999
Abstract The conformational response of calf thymus DNA to solvent conditions altered by varying amounts of ethanol, 2-propanol and tert-butanol has been monitored by circular dichroism. These measurements reveal above a critical cosolvent mole fraction x*2 typical for each alcohol, a condensed form of the macromolecule with unusually large ellipticity in the 250–300 nm region similar to the well-known c( − ) forms induced by above-critical concentrations of neutral polymers and salt. This alcohol induced transition is completely reversible and it appears in the concentration range (x*=0.135, 0.076, 0.055 for ethanol, 2-propanol and tert-butanol, respectively) where change in 2 the association state of the alcohol molecules occurs as inferred from compressibility and IR data. Both DNA condensation and alcohol association processes start when almost all water is involved in hydration structures. The present data clearly indicate the important role that water and solvation play in the self-association of alcohol molecules and in the conformation of nucleic acids. © 1999 Elsevier Science B.V. All rights reserved. Keywords: DNA condensation; Water/alcohol mixtures; Circular dichroism; Adiabatic compressibility
1. Introduction Addition of certain solutes or solvents to DNA converts the molecule from its extended conformation to compact toroids, rods or spheres [1]. Compact structures of DNA are of biological interest because within viruses and cells DNA generally exists in a highly compact, condensed state. Many different types of substances including multivalent cations such as polyamines, basic proteins, cationic liposomes can induce condensation in vitro. Even neutral polymers, such as polyethylene * Corresponding author.
glycol (PEG) and alcohols, at high concentration of salts can induce DNA aggregation into structures with distinctive morphology and optical rotation properties. This phenomenon is called polymer-and-salt-induced (c) condensation. c-Type compaction is distinguished by its dramatically altered circular dichroism (CD) spectrum and the extremely enhanced magnitude of ellipticity. The c-type condensed DNA can give CD spectra with either positive, c(+ ), or negative, c(− ), ellipticity maximum near 280 nm. Both c(+ ) and c(− ) conformational transitions of DNA have been observed by above-critical concentration of ethanol and salt [2].
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DNA condensation has been observed by a variety of techniques [1] including electron microscopy, dynamic light scattering, sedimentation, viscosimetry. Very recently fluorescence microscopy was used to observe the condensation of single large T4 DNA molecule in PEG [3,4] and in 2-propanol [5] solutions. These results provide definite evidence that an individual DNA chain exhibits a first order phase transition between an elongated coil and a compact globule with a change in the concentration of PEG or 2propanol added to the aqueous environment. Such kind of conformational changes represents an example of a polymer coil – globule transition (well known to polymer physicists) in which the polymer chain may pass from one state to another changing the quality of the solvent [6]. DNA condensation arises from a complex interplay of interactions and the factor that control and modulate these high-order conformational modifications are far from being fully understood. Wilson and Bloomfield [7] showed that DNA condenses under a variety of ionic conditions when approximately 90% of its charge is neutralised by counterion condensation. Theoretical estimates of the various contributions to the free energy of condensation also implicated electrostatics as the major factor [8,9]. However, other studies have suggested that factors other than electrostatics are dominant in the condensation of DNA molecules. Rau et al. [10] suggest that water structure in the interhelical space is the determinant factor of the condensation. The specific effects of different condensing agents are attributed to their different effects on water structure. In the last years our group has been interested in studying the role of the solvent in maintenance of macromolecular native state and in self-assembly processes. The approach is to alter the composition of the solvent by adding little quantities of monohydric alcohols and to study the induced changes in the process under examination as a function of alcohol concentration. The aim is to establish correlations between the effects of alcohols on the conformational and dynamical properties of biomolecules and changes of properties of the solvent.
The mechanism of action of alcohols on biomolecules is not clear and there are different views considering this action as a ligand binding or as an indirect effect, involving changes in the properties of solvent water [11,12]. In previous papers the effects of monohydric alcohols on very different systems and process, i.e. the micellization of surfactant molecules, [12–14] the thermal denaturation of transfer-ribonucleic acids [12,15] and the thermal unfolding of proteins, [16] were shown as strikingly similar and closely connected to specific properties of alcohol/water mixtures [17]. Accordingly, these results support the idea that the observed effects are not due to a binding of alcohol molecules to groups in macromolecules, but to an indirect mechanism related to changes in the solvent properties caused by alcohols. The question arises on which of the physical parameters of the solvent are crucial for the observed effects. On this regard, the effect of alcohol, often considered as a means for lowering the dielectric constant of solvent and to affect the interactions among charged groups appears more complex. Our data cannot be accounted for satisfactorily by considering the solvent as a continuous characterised by bulk properties such as the dielectric constant; instead the structure of the solvent and structural changes that take place in the hydration regions appears to play a dominant role in the observed processes. Available literature data [11] provide further support that the same conclusion could be valid for a number of other systems of biological and chemical interest. In this paper, in continuation of our previous work, the conformational response of calf thymus DNA to solvent conditions altered by varying amounts of ethanol, 2-propanol and tert-butanol has been monitored by circular dichroism. The measurements reveal above a critical alcohol concentration, typical for each alcohol, a condensed form of the macromolecule with unusually large positive or negative ellipticity similar to the wellknown c forms. The results are discussed in connection with previous adiabatic compressibility data of water/alcohol mixtures in the same concentration range [18]. The main object of the present investigation is to extend information about the influence of the solvent on the c condensation process of DNA molecules.
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2. Material and methods Calf thymus DNA (Sigma product) was dissolved in 0.8 M NaCl, 0.1 mM EDTA at pH 7 and sonicated to reduce the viscosity for 30 s with a Soniprep 150 ultrasonic disintegrator. The solutions were prepared by weighing, mixing a fixed amount of the DNA solution with water/alcohol mixtures (0.8 M NaCl, pH 7) previously prepared at the desired molar fraction of alcohol. DNA concentrations (67 mM of nucleotide phosphate) were determined by using a Cary 2290 spectrophotometer and extinction coefficient at 260 nm of 6600 M − 1 cm − 1 [2]. CD spectra between 210 and 320 nm were taken on a Jasco 710 spectropolarimeter using cells with a 1 cm light path. All CD spectra were recorded with the sample compartment thermostated at 25°C. 3. Results and discussion The physical properties of nucleic acids and proteins in alcohol/water mixtures have been investigated in a number of laboratories [11]. Such studies, it is believed, allow assessments of the relative importance of different types of interactions in conferring stability of these polymers in solution. This, in particular, can appear by comparing data from alcohols having hydrophobic groups of different sizes. In ethanol, 2-propanol, tert-butanol series a hydrophobic group of increasing size is present and it can be regarded as obtained by successive substitution of an − H atom in a methanol molecule by a −CH3 group. The role of the salt in a DNA solution is to overcome the electrostatic repulsive force within the DNA chain by counterion binding. Condensation is impossible in solution of low ionic strength since it is impeded by repulsion of the charges of the phosphate groups. We have, therefore, considered the case of a very high NaCl concentration (0.8 M).
3.1. DNA condensation in water/alcohol mixtures The CD spectra of calf thymus DNA in solutions of various ethanol, 2-propanol and tert-butanol contents are given in Fig. 1.
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3.1.1. Ethanol In the 0–0.125 ethanol mole fraction (x2) range the spectrum shows a maximum at about 275 nm, a zero point at 257 nm, and a minimum centred around 247 nm. The maximum molar ellipticity [u] at 275 nm is 6.6·103 deg cm − 2 per dmol. These features characterise the CD spectrum B-form of DNA [19]. In 0.140 ethanol mole fraction, however, the spectrum is dramatically different. It has a very large positive maximum around 270–275 nm similar to the well known c(+) form. From x2 \ 0.140 the shape of the spectra is similar to that at 0.140 but the overall magnitude is diminished. 3.1.2. 2 -Propanol In the 0–0.071 2-propanol mole fraction range the spectrum is that of B-form of DNA. The spectrum at 0.075 2-propanol mole fraction has a large minimum with negative ellipticity between 250 and 300 nm characteristic of the familiar c(− ) spectrum. A mere increase in 2-propanol concentration at x2 = 0.078 converts such c(−) sample into c(+) states having anomalous large positive ellipticity around 270 nm. On increasing x2 the magnitude of the spectra diminished. 3.1.3. tert-Butanol In the 0–0.050 tert-butanol mole fraction range the spectrum is that of B-form of DNA. At 0.062 mole fraction tert-butanol gives a spectrum with a large minimum with negative ellipticity between 250 and 300 nm similar to the well-known c(−) forms produced by above critical concentrations of poly-(ethylene oxide) and salt [20,21]. Fig. 2 shows [u]275 as a function of x2 for the three alcohols under study. A sharp transition is observed centred around x*= 0.135, 0.076, 0.055, 2 for ethanol, 2-propanol and tert-butanol water mixtures, respectively. This alcohol induced transition is completely reversible and highly co-operative, in that it occurs over a very small concentration range (Dx2 0.01) of cosolvent. The transition occurs at lower x2 values as the hydrophobic group becomes larger, suggesting an important role of hydrophobic effect in DNA condensation.
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c Condensation of calf thymus DNA in ethanol/NaCl aqueous solutions has been previously studied in detail [2]. These measurements reveal, above critical concentrations of ethanol and salt, a condensed form of the macromolecule with unusually large positive or large negative ellipticity. Mere increase in NaCl concentration at constant concentration of ethanol suffices to convert c( + ) into c( −) state. It is difficult to understand the importance of environmental factors in determining c enantiomorphism. Our data show a greater tendency to the c( −) form as the hydrophobicity of alcohol is increased. Direct observation of a single DNA molecule with fluorescence microscopy is the best way to study the globule state of macromolecule. It is recently found by fluorescence microscopy that a single DNA chain changes its higher order structure between expanded coil and shrunken globule in a discrete manner with a change in the concentration of 2-propanol added to the aqueous environment [5]. The transition between the coil and globule is observed at a concentration of around 30% in volume of 2-propanol, the same concentration value where the transition in [u]275 is observed (Fig. 2). No literature data on the DNA structure in water/tert-butanol mixtures were found.
3.2. Compressibility measurements Our data indicate an important role of alcohol hydrophobicity on DNA condensation and suggest that other factors, in addition to the electrostatic ones, are at work in this process. On this regard it is to note that this alcohol induced transition occur in the same concentration range where an anomalous behaviour in several properties of water/alcohol mixtures themselves is also observed; in this region of composition a maximum is found in sound absorption, [22] X-ray scattering [23] and light scattering [24,25]. Our recent studies [18] on properties of water/alcohol Fig. 1. CD spectra of calf thymus DNA at various alcohol concentrations. Ethanol: (—): 0 5 x2 5 0.125; (---): x2 =0.140; (···): x2 = 0.167; (-·-): x2 = 0.197. 2-Propanol: (—): 0 5x2 5 0.071; (-··-): x2 = 0.075; (---): x2 = 0.078; (···): x2 = 0.082; (-·-): x2 = 0.111. Tert-butanol: (—): 05 x2 5 0.050; (-·-): x2 =0.056; (···): x2 = 0.062; (---): x2 = 0.074.
Fig. 1.
M. Matzeu et al. / Colloids and Surfaces B: Biointerfaces 13 (1999) 157–163
mixtures show that this anomalous behaviour can be associated to some kind of ‘hydrophobic clustering’ of alcohol molecules in the water rich region of composition beyond a threshold value of alcohol concentration. Plots of alcohol apparent molar compressibility, Fk versus x2 at 25°C are reported in Fig. 3. This quantity is nearly constant at low alcohol concentration, but it increases steeply as more alcohol is added to the solution. The compressibility data have been discussed in reference [18] by considering three mole fractions ranges defined by ‘signpost’ mole fractions x2a and x2b. It is found that x2a and x2b values shift to lower concentrations as the hydrophobic group of alcohol molecules becomes larger. The data suggest that in the 0 to x2a range, where Fk do not change with x2, the alcohol molecules are essentially dispersed and surrounded by ‘water cages’ of fairly regular and longer-lived H bonds. In the x2a – x2b region a transition attributed to a progressive clustering of alcohol molecules with strong modification in the solvation of hydrophobic group is observed. In this concentration range a qualitative change in the nature of the interactions between solvent components occurs [16]. Interactions between hydrophobic groups become increasingly favourable and progressively replace the interactions of these groups with water molecules. In the water/DNA/ alcohol systems a modification in the water/DNA and alcohol/DNA interactions in this concentration range is also expected [16]. Actually, the first derivative of Fk (Fig. 3) displays a maximum at x2 =0.143, 0.071, 0.053 for ethanol, 2-propanol and tert-butanol/water mixtures, respectively. These values localise the transition in the x2a to x2b range [26] and are practically coincident with the values x*2 where the conformational transition of DNA is observed. A correlation between the effect of alcohols on the conformational properties of DNA and changes in properties of the solvent clearly appears from the data. An approximate evaluation of the number of water molecules located in the first hydration layer of an alcohol molecule shows that the self association of alcohol molecules starts when all the water is involved in hydration structures [17]. This suggests that water and solvation plays a dominant role in the process of self-association of alcohol
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Fig. 2. Molar ellipticity [u] at 275 nm as a function of mole fraction of alcohol.
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molecules and in the c conformational transition of DNA. This point of view is in line with the Rau and Parsegian proposal [10] that the driving force for DNA condensation is due to intervening water structure. Previous results [16,17] suggest that addition of short alcohols affects the micellization of surfactant molecules as well as the thermal unfolding of ribonucleic acids and proteins, modifying the extent of enthalpy and entropy contributions associated with structural reorganisation of water in these processes. In other works, it seems that alcohols, at low concentration, essentially affect hydrophobic interactions. In reference [16] it has been suggested, consistently with Shinoda’s point of view, [27] that hydrophobic interactions at low concentrations of hydrophobic groups are repulsive and disfavours aggregation of non-polar species. Therefore, the attenuation of these interactions, due to alcohol addition, should favour clustering of hydrophobic groups and more compact structures. Present results on condensation of DNA by monohydric alcohols are consistent with previous work [16,17] and indicate that the total mixing scheme of the binary solvent mixture, including the possibility of clustering effects, can have a dramatic influence on DNA conformation.
Acknowledgements This work was supported in part from contribution of the Consiglio Nazionale delle Ricerche (Rome).
References
Fig. 3. Plots of alcohol apparent molar compressibility, Fk (), and the first derivative Fk () in water/alcohol mixtures versus x2.
[1] A. Bloomfield, Curr. Opin. Struct. Biol. 6 (1996) 334. [2] R. Huey, S.C. Mohr, Biopolymers 20 (1981) 2533. [3] K. Minagawa, Y. Matsuzawa, K. Yoshikawa, A.R. KhokUlov, M. Doi, Biopolymers 34 (1994) 555. [4] K. Yoshikawa, Y. Matsuzawa, Physica D 84 (1995) 220. [5] M. Ueda, K. Yoshikawa, Phys. Rev. Lett. 77 (1996) 2133. [6] I.M. Lifshitz, A.Y. Grosberg, A.R. Khokhlov, Rev. Mod. Phys. 50 (1978) 683. [7] R.W. Wilson, V.A. Bloomfield, Biochemistry 18 (1979) 2192. [8] R. Marquet, C. Houssier, J. Biol. Struct. Dyn. 9 (1991) 159. [9] S.C. Riemer, V.A. Bloomfield, Biopolymers 17 (1978) 785.
M. Matzeu et al. / Colloids and Surfaces B: Biointerfaces 13 (1999) 157–163 [10] D.C. Rau, V.A. Parsegian, Biophys. J. 61 (1992) 246. [11] D. Eagland, in: F. Franks (Ed.), Water: A Comprehensive Treatise, vol. 4, Plenum, New York, 1975, p. 305. [12] G. Onori, S. Passeri, A. Cipiciani, J. Phys. Chem. 93 (1989) 4306. [13] A. Cipiciani, G. Onori, G. Savelli, Chem. Phys. Lett. 143 (1988) 505. [14] G. Onori, A. Santucci, Chem. Phys. Lett. 189 (1992) 598. [15] S. Beneventi, G. Onori, Biophys. Chem. 25 (1986) 181. [16] S. Cinelli, G. Onori, A. Santucci, J. Phys. Chem. B 101 (1997) 8029. [17] G. Onori, A. Santucci, J. Mol. Liquids 69 (1996) 161. [18] M. D’Angelo, G. Onori, A. Santucci, J. Chem. Phys. 100 (1994) 3107.
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[19] M.J.B. Tunis-Schneider, M.F. Maestre, J. Mol. Biol. 52 (1970) 521. [20] S.M. Cheng, S.C. Mohr, FEBS Lett. 49 (1974) 37. [21] S.M. Cheng, S.C. Mohr, Biopolymers 14 (1975) 663. [22] M.J. Blandamer, Advances in Physical Organic Chemistry, vol. 2;, Academic Press, New York, 1973, p. 495. [23] H.D. Bale, R.E. Shepler, P. Sorgen, Phys. Chem. Liquids 1 (1970) 181. [24] N. Ito, T. Fujiama, Y. Udagawa, Bull. Chem. Soc. Jpn. 56 (1983) 379. [25] M.F. Vuks, L.V. Shurupova, Opt. Spectrosk. 40 (1976) 86. [26] G. Onori, A. Santucci, J. Phys. Chem. B 101 (1997) 4662. [27] K.J. Shinoda, J. Phys. Chem. 81 (1977) 1300.
Condensation of DNA by monohydric alcohols M. Matzeu a, G. Onori b,*, A. Santucci b b
a Istituto Superiore di Sanita`, Laboratorio di Fisica, V.le Regina Elena, I-00161 Roma, Italy Dipartimento di Fisica, Istituto per la Fisica della Materia, Unita` di Perugia, Uni6ersita` di Perugia, Via A. Pascoli, I-06100 Perugia, Italy
Accepted 3 February 1999
Abstract The conformational response of calf thymus DNA to solvent conditions altered by varying amounts of ethanol, 2-propanol and tert-butanol has been monitored by circular dichroism. These measurements reveal above a critical cosolvent mole fraction x*2 typical for each alcohol, a condensed form of the macromolecule with unusually large ellipticity in the 250–300 nm region similar to the well-known c( − ) forms induced by above-critical concentrations of neutral polymers and salt. This alcohol induced transition is completely reversible and it appears in the concentration range (x*=0.135, 0.076, 0.055 for ethanol, 2-propanol and tert-butanol, respectively) where change in 2 the association state of the alcohol molecules occurs as inferred from compressibility and IR data. Both DNA condensation and alcohol association processes start when almost all water is involved in hydration structures. The present data clearly indicate the important role that water and solvation play in the self-association of alcohol molecules and in the conformation of nucleic acids. © 1999 Elsevier Science B.V. All rights reserved. Keywords: DNA condensation; Water/alcohol mixtures; Circular dichroism; Adiabatic compressibility
1. Introduction Addition of certain solutes or solvents to DNA converts the molecule from its extended conformation to compact toroids, rods or spheres [1]. Compact structures of DNA are of biological interest because within viruses and cells DNA generally exists in a highly compact, condensed state. Many different types of substances including multivalent cations such as polyamines, basic proteins, cationic liposomes can induce condensation in vitro. Even neutral polymers, such as polyethylene * Corresponding author.
glycol (PEG) and alcohols, at high concentration of salts can induce DNA aggregation into structures with distinctive morphology and optical rotation properties. This phenomenon is called polymer-and-salt-induced (c) condensation. c-Type compaction is distinguished by its dramatically altered circular dichroism (CD) spectrum and the extremely enhanced magnitude of ellipticity. The c-type condensed DNA can give CD spectra with either positive, c(+ ), or negative, c(− ), ellipticity maximum near 280 nm. Both c(+ ) and c(− ) conformational transitions of DNA have been observed by above-critical concentration of ethanol and salt [2].
0927-7765/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 6 5 ( 9 9 ) 0 0 0 0 5 - 3
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DNA condensation has been observed by a variety of techniques [1] including electron microscopy, dynamic light scattering, sedimentation, viscosimetry. Very recently fluorescence microscopy was used to observe the condensation of single large T4 DNA molecule in PEG [3,4] and in 2-propanol [5] solutions. These results provide definite evidence that an individual DNA chain exhibits a first order phase transition between an elongated coil and a compact globule with a change in the concentration of PEG or 2propanol added to the aqueous environment. Such kind of conformational changes represents an example of a polymer coil – globule transition (well known to polymer physicists) in which the polymer chain may pass from one state to another changing the quality of the solvent [6]. DNA condensation arises from a complex interplay of interactions and the factor that control and modulate these high-order conformational modifications are far from being fully understood. Wilson and Bloomfield [7] showed that DNA condenses under a variety of ionic conditions when approximately 90% of its charge is neutralised by counterion condensation. Theoretical estimates of the various contributions to the free energy of condensation also implicated electrostatics as the major factor [8,9]. However, other studies have suggested that factors other than electrostatics are dominant in the condensation of DNA molecules. Rau et al. [10] suggest that water structure in the interhelical space is the determinant factor of the condensation. The specific effects of different condensing agents are attributed to their different effects on water structure. In the last years our group has been interested in studying the role of the solvent in maintenance of macromolecular native state and in self-assembly processes. The approach is to alter the composition of the solvent by adding little quantities of monohydric alcohols and to study the induced changes in the process under examination as a function of alcohol concentration. The aim is to establish correlations between the effects of alcohols on the conformational and dynamical properties of biomolecules and changes of properties of the solvent.
The mechanism of action of alcohols on biomolecules is not clear and there are different views considering this action as a ligand binding or as an indirect effect, involving changes in the properties of solvent water [11,12]. In previous papers the effects of monohydric alcohols on very different systems and process, i.e. the micellization of surfactant molecules, [12–14] the thermal denaturation of transfer-ribonucleic acids [12,15] and the thermal unfolding of proteins, [16] were shown as strikingly similar and closely connected to specific properties of alcohol/water mixtures [17]. Accordingly, these results support the idea that the observed effects are not due to a binding of alcohol molecules to groups in macromolecules, but to an indirect mechanism related to changes in the solvent properties caused by alcohols. The question arises on which of the physical parameters of the solvent are crucial for the observed effects. On this regard, the effect of alcohol, often considered as a means for lowering the dielectric constant of solvent and to affect the interactions among charged groups appears more complex. Our data cannot be accounted for satisfactorily by considering the solvent as a continuous characterised by bulk properties such as the dielectric constant; instead the structure of the solvent and structural changes that take place in the hydration regions appears to play a dominant role in the observed processes. Available literature data [11] provide further support that the same conclusion could be valid for a number of other systems of biological and chemical interest. In this paper, in continuation of our previous work, the conformational response of calf thymus DNA to solvent conditions altered by varying amounts of ethanol, 2-propanol and tert-butanol has been monitored by circular dichroism. The measurements reveal above a critical alcohol concentration, typical for each alcohol, a condensed form of the macromolecule with unusually large positive or negative ellipticity similar to the wellknown c forms. The results are discussed in connection with previous adiabatic compressibility data of water/alcohol mixtures in the same concentration range [18]. The main object of the present investigation is to extend information about the influence of the solvent on the c condensation process of DNA molecules.
M. Matzeu et al. / Colloids and Surfaces B: Biointerfaces 13 (1999) 157–163
2. Material and methods Calf thymus DNA (Sigma product) was dissolved in 0.8 M NaCl, 0.1 mM EDTA at pH 7 and sonicated to reduce the viscosity for 30 s with a Soniprep 150 ultrasonic disintegrator. The solutions were prepared by weighing, mixing a fixed amount of the DNA solution with water/alcohol mixtures (0.8 M NaCl, pH 7) previously prepared at the desired molar fraction of alcohol. DNA concentrations (67 mM of nucleotide phosphate) were determined by using a Cary 2290 spectrophotometer and extinction coefficient at 260 nm of 6600 M − 1 cm − 1 [2]. CD spectra between 210 and 320 nm were taken on a Jasco 710 spectropolarimeter using cells with a 1 cm light path. All CD spectra were recorded with the sample compartment thermostated at 25°C. 3. Results and discussion The physical properties of nucleic acids and proteins in alcohol/water mixtures have been investigated in a number of laboratories [11]. Such studies, it is believed, allow assessments of the relative importance of different types of interactions in conferring stability of these polymers in solution. This, in particular, can appear by comparing data from alcohols having hydrophobic groups of different sizes. In ethanol, 2-propanol, tert-butanol series a hydrophobic group of increasing size is present and it can be regarded as obtained by successive substitution of an − H atom in a methanol molecule by a −CH3 group. The role of the salt in a DNA solution is to overcome the electrostatic repulsive force within the DNA chain by counterion binding. Condensation is impossible in solution of low ionic strength since it is impeded by repulsion of the charges of the phosphate groups. We have, therefore, considered the case of a very high NaCl concentration (0.8 M).
3.1. DNA condensation in water/alcohol mixtures The CD spectra of calf thymus DNA in solutions of various ethanol, 2-propanol and tert-butanol contents are given in Fig. 1.
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3.1.1. Ethanol In the 0–0.125 ethanol mole fraction (x2) range the spectrum shows a maximum at about 275 nm, a zero point at 257 nm, and a minimum centred around 247 nm. The maximum molar ellipticity [u] at 275 nm is 6.6·103 deg cm − 2 per dmol. These features characterise the CD spectrum B-form of DNA [19]. In 0.140 ethanol mole fraction, however, the spectrum is dramatically different. It has a very large positive maximum around 270–275 nm similar to the well known c(+) form. From x2 \ 0.140 the shape of the spectra is similar to that at 0.140 but the overall magnitude is diminished. 3.1.2. 2 -Propanol In the 0–0.071 2-propanol mole fraction range the spectrum is that of B-form of DNA. The spectrum at 0.075 2-propanol mole fraction has a large minimum with negative ellipticity between 250 and 300 nm characteristic of the familiar c(− ) spectrum. A mere increase in 2-propanol concentration at x2 = 0.078 converts such c(−) sample into c(+) states having anomalous large positive ellipticity around 270 nm. On increasing x2 the magnitude of the spectra diminished. 3.1.3. tert-Butanol In the 0–0.050 tert-butanol mole fraction range the spectrum is that of B-form of DNA. At 0.062 mole fraction tert-butanol gives a spectrum with a large minimum with negative ellipticity between 250 and 300 nm similar to the well-known c(−) forms produced by above critical concentrations of poly-(ethylene oxide) and salt [20,21]. Fig. 2 shows [u]275 as a function of x2 for the three alcohols under study. A sharp transition is observed centred around x*= 0.135, 0.076, 0.055, 2 for ethanol, 2-propanol and tert-butanol water mixtures, respectively. This alcohol induced transition is completely reversible and highly co-operative, in that it occurs over a very small concentration range (Dx2 0.01) of cosolvent. The transition occurs at lower x2 values as the hydrophobic group becomes larger, suggesting an important role of hydrophobic effect in DNA condensation.
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c Condensation of calf thymus DNA in ethanol/NaCl aqueous solutions has been previously studied in detail [2]. These measurements reveal, above critical concentrations of ethanol and salt, a condensed form of the macromolecule with unusually large positive or large negative ellipticity. Mere increase in NaCl concentration at constant concentration of ethanol suffices to convert c( + ) into c( −) state. It is difficult to understand the importance of environmental factors in determining c enantiomorphism. Our data show a greater tendency to the c( −) form as the hydrophobicity of alcohol is increased. Direct observation of a single DNA molecule with fluorescence microscopy is the best way to study the globule state of macromolecule. It is recently found by fluorescence microscopy that a single DNA chain changes its higher order structure between expanded coil and shrunken globule in a discrete manner with a change in the concentration of 2-propanol added to the aqueous environment [5]. The transition between the coil and globule is observed at a concentration of around 30% in volume of 2-propanol, the same concentration value where the transition in [u]275 is observed (Fig. 2). No literature data on the DNA structure in water/tert-butanol mixtures were found.
3.2. Compressibility measurements Our data indicate an important role of alcohol hydrophobicity on DNA condensation and suggest that other factors, in addition to the electrostatic ones, are at work in this process. On this regard it is to note that this alcohol induced transition occur in the same concentration range where an anomalous behaviour in several properties of water/alcohol mixtures themselves is also observed; in this region of composition a maximum is found in sound absorption, [22] X-ray scattering [23] and light scattering [24,25]. Our recent studies [18] on properties of water/alcohol Fig. 1. CD spectra of calf thymus DNA at various alcohol concentrations. Ethanol: (—): 0 5 x2 5 0.125; (---): x2 =0.140; (···): x2 = 0.167; (-·-): x2 = 0.197. 2-Propanol: (—): 0 5x2 5 0.071; (-··-): x2 = 0.075; (---): x2 = 0.078; (···): x2 = 0.082; (-·-): x2 = 0.111. Tert-butanol: (—): 05 x2 5 0.050; (-·-): x2 =0.056; (···): x2 = 0.062; (---): x2 = 0.074.
Fig. 1.
M. Matzeu et al. / Colloids and Surfaces B: Biointerfaces 13 (1999) 157–163
mixtures show that this anomalous behaviour can be associated to some kind of ‘hydrophobic clustering’ of alcohol molecules in the water rich region of composition beyond a threshold value of alcohol concentration. Plots of alcohol apparent molar compressibility, Fk versus x2 at 25°C are reported in Fig. 3. This quantity is nearly constant at low alcohol concentration, but it increases steeply as more alcohol is added to the solution. The compressibility data have been discussed in reference [18] by considering three mole fractions ranges defined by ‘signpost’ mole fractions x2a and x2b. It is found that x2a and x2b values shift to lower concentrations as the hydrophobic group of alcohol molecules becomes larger. The data suggest that in the 0 to x2a range, where Fk do not change with x2, the alcohol molecules are essentially dispersed and surrounded by ‘water cages’ of fairly regular and longer-lived H bonds. In the x2a – x2b region a transition attributed to a progressive clustering of alcohol molecules with strong modification in the solvation of hydrophobic group is observed. In this concentration range a qualitative change in the nature of the interactions between solvent components occurs [16]. Interactions between hydrophobic groups become increasingly favourable and progressively replace the interactions of these groups with water molecules. In the water/DNA/ alcohol systems a modification in the water/DNA and alcohol/DNA interactions in this concentration range is also expected [16]. Actually, the first derivative of Fk (Fig. 3) displays a maximum at x2 =0.143, 0.071, 0.053 for ethanol, 2-propanol and tert-butanol/water mixtures, respectively. These values localise the transition in the x2a to x2b range [26] and are practically coincident with the values x*2 where the conformational transition of DNA is observed. A correlation between the effect of alcohols on the conformational properties of DNA and changes in properties of the solvent clearly appears from the data. An approximate evaluation of the number of water molecules located in the first hydration layer of an alcohol molecule shows that the self association of alcohol molecules starts when all the water is involved in hydration structures [17]. This suggests that water and solvation plays a dominant role in the process of self-association of alcohol
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Fig. 2. Molar ellipticity [u] at 275 nm as a function of mole fraction of alcohol.
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molecules and in the c conformational transition of DNA. This point of view is in line with the Rau and Parsegian proposal [10] that the driving force for DNA condensation is due to intervening water structure. Previous results [16,17] suggest that addition of short alcohols affects the micellization of surfactant molecules as well as the thermal unfolding of ribonucleic acids and proteins, modifying the extent of enthalpy and entropy contributions associated with structural reorganisation of water in these processes. In other works, it seems that alcohols, at low concentration, essentially affect hydrophobic interactions. In reference [16] it has been suggested, consistently with Shinoda’s point of view, [27] that hydrophobic interactions at low concentrations of hydrophobic groups are repulsive and disfavours aggregation of non-polar species. Therefore, the attenuation of these interactions, due to alcohol addition, should favour clustering of hydrophobic groups and more compact structures. Present results on condensation of DNA by monohydric alcohols are consistent with previous work [16,17] and indicate that the total mixing scheme of the binary solvent mixture, including the possibility of clustering effects, can have a dramatic influence on DNA conformation.
Acknowledgements This work was supported in part from contribution of the Consiglio Nazionale delle Ricerche (Rome).
References
Fig. 3. Plots of alcohol apparent molar compressibility, Fk (), and the first derivative Fk () in water/alcohol mixtures versus x2.
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